JP2015527350A - Factor VIII liquid formulation - Google Patents

Abstract

The present invention provides a liquid aqueous formulation of coagulation factor VIII comprising a factor VIII molecule, a calcium salt at a concentration of greater than 10 mM, and a saccharide and / or polyol at a concentration of at least 100 mM and having a pH of 5.5 to 7.5. Target. The present invention is a method for optimizing a liquid formulation of coagulation factor VIII, comprising: (i) providing one or more liquid formulations comprising factor VIII to be tested; and (ii) Adding a protein denaturant to the liquid formulation and incubating the resulting solution for a predetermined period of time; and (iii) analyzing the incubated solution of (ii) for the presence of dissociated factor VIII; and iv) selecting one or more formulations having the desired low level of dissociated factor VIII.

Description

  In subjects with coagulation disorders, such as humans with hemophilia A, the various steps of the coagulation cascade fail, for example, due to the absence or insufficient presence of coagulation factors. Such dysfunction of part of the coagulation cascade results in inadequate blood clotting and damage to viscera such as bleeding or joints that can be life-threatening. Individuals with hemophilia A can receive coagulation factor replacement therapy, such as exogenous factor VIII (FVIII).

  All Factor VIII products currently on the market are supplied as freeze-dried preparations for intravenous infusion. The person performing the injection needs to reconstitute the powder with the liquid before use. This operation is time consuming and can be a multi-step manual operation, requiring the user to visually monitor dissolution, which can take several minutes and can cause some dose variation . Thus, a stable liquid ready-to-use formulation of factor VIII molecules (preferably combined with a convenient delivery system) is highly desirable. Liquid formulations with very high concentrations of stabilizing excipients can cause local hypersensitivity and possibly phlebitis at the site of injection due to large differences in osmotic pressure between the injected liquid and the surrounding tissue. Therefore, a liquid formulation having an osmotic concentration close to physiological conditions is particularly desirable.

  Proteins have many possible degradation pathways in aqueous solution. Therefore, the liquid protein preparation needs to be precisely adjusted in order to obtain stability in the storage period required for practical use. The shelf life typically required to allow production, storage and distribution is 2 years at a storage temperature of 2-8 ° C. In the process of discovering excipients, pH values and other possible factors that can improve stability in liquid formulations, the sample needs to be incubated for a very long time to observe the assumed stabilization effect It is impractical to use only targeted storage conditions. Thus, stability tests are often performed under accelerated (stress) conditions. Such stress conditions include, for example, high temperature, high humidity, strong light, extreme pH, an increase in the gas-liquid interface by vortexing or shaking, and / or repeated freeze-thaw cycles. Due to such stress conditions, whether or not the data obtained from the accelerated stability test can be extrapolated to the data under real-time conditions and how much can be extrapolated is an important issue.

  Therefore, a method for performing a reliable stability test that avoids long-term storage and avoids stress conditions is highly desirable.

  The stability of factor VIII liquid formulations has been previously described in the academic literature and in patents and patent applications.

  Fatouros et al [International Journal of Pharmaceutics 155, 121-131 (1997)] describe the effects of oxygen, metal ions, pH and ionic strength on the stability of B domain-deficient factor VIII in liquid solutions. Part of this work is also described in US5962650.

  Fatouros et al. [Pharmaceutical Research 14, 1679-1684 (1997) and International Journal of Pharmaceutics 194, 69-79 (2000)] describe the stabilizing effects of surfactants and especially very high concentrations of carbohydrates. Yes. Part of this work is also described in US5919908.

  WO2011 / 027152 describes a liquid formulation of factor VIII, buffered by a combination of tris and potassium benzoate, containing EDTA and calcium, excess calcium relative to EDTA, and additional excipients. ing.

US5962650 US5919908 WO2011 / 027152 US20100261872 WO2010045568 WO2009062100 WO2010014708 WO2008082669 WO2007126808 US20100173831 US20100173830 US20100168391 US20100113365 US20100113364 WO200331464 WO2009108806 WO2010102886 WO2010115866 WO2011101242 (PCT / EP2011 / 051438) WO2011101284 (PCT / EP2011 / 051959) WO2011101277 (PCT / EP2011 / 051889) WO2011131510 (PCT / EP2011 / 055686) WO2012007324 (PCT / EP2011 / 061349) WO2011101267 (PCT / EP2011 / 051723) WO2013083858 WO2008 / 135501 WO2009 / 007451

Fatouros et al. [International Journal of Pharmaceutics 155, 121-131 (1997)] Fatouros et al. [Pharmaceutical Research 14, 1679-1684 (1997)] Fatouros et al. [International Journal of Pharmaceutics 194, 69-79 (2000)] McCormick, C.L., A.B.Lowe, and N. Ayres, Water-Soluble Polymers, Encyclopedia of Polymer Science and Technology. 2002, John Wiley & Sons, Inc. Remington: The Science and Practice of Pharmacy, 19th edition, 1995 Thim L. et al. (Haemophilia 2010; 16: 349-359) European Pharmacopoeia 2.2.35 United States Pharmacopeia Chapter 785

  The present invention relates to a liquid pharmaceutical formulation of FVIII that can be used to treat a subject with hemophilia A.

  Therefore, the present inventors combined a calcium concentration of more than 10 mM, for example 15 mM or more, and a concentration of polyol of 100 mM or more for a factor VIII molecule containing a derivative such as a PEGylated factor VIII molecule having a delayed action. Has been found to improve the stability of liquid formulations at refrigerated temperatures. Due to the high stabilizing effect of these concentrations of calcium and certain polyols, stable liquid formulations having low concentrations of NaCl and low osmotic pressure concentrations can be obtained.

  Thus, in one aspect, the invention comprises a Factor VIII molecule, one or more calcium salts that provide a concentration of calcium ions greater than 10 mM, and one or more polyols at a concentration of at least 100 mM, Of interest are liquid aqueous formulations of coagulation factor VIII having a pH in the range of -7.5.

  In one embodiment, the present invention relates to a coagulation factor VIII comprising a factor VIII molecule, a calcium salt at a concentration of at least 15 mM, and a saccharide and / or polyol at a concentration of at least 100 mM and having a pH of 5.5 to 7.5. For liquid aqueous formulations.

  Therefore, the present inventors have further discovered that the stabilizing effect of the excipient in the liquid preparation of polyvalent protein can be confirmed accurately by an accelerated assay in which the sample is incubated in a short time including a chemical protein denaturant. .

  Thus, in another aspect, the invention provides a method for optimizing a liquid formulation of coagulation factor VIII and identifying a stable liquid formulation of factor VIII comprising: (i) FVIII and in FVIII Providing one or more liquid formulations comprising one or more excipients to be evaluated for stabilizing effect; and (ii) a protein denaturant selected for the liquid formulation (e.g., guanidinium chloride). Or (urea) and incubating the resulting solution for a predetermined period of time; (iii) analyzing the incubated solution of (ii) for the presence of dissociated factor VIII; (iv) desired Selecting one or more formulations having low levels of dissociated factor VIII.

  The stable liquid pharmaceutical formulation of the FVIII molecule of the present invention facilitates the use of an improved (easy to use) delivery system. The stabilization principle described can also be used to stabilize factor VIII molecules in manufacturing (upstream purification steps, storage and handling).

Coagulation factor VIII molecule Factor VIII (FVIII) is a large complex glycoprotein produced primarily by hepatocytes. FVIII has a size of approximately 300 kDa, including the signal peptide, and contains several distinct domains defined by homology. There are 3 A-domains, a unique B domain and 2 C-domains. Small acidic region C-terminus of A1 (a1 region) and A2 (a2 region) and N-terminus of A3 domain (a3 region) include thrombin and von Willebrand factor (vWF or VWF), FVIII carrier protein, etc. Plays an important role in the interaction of clotting proteins with itself. Therefore, the detailed domain structure can be described as A1-a1-A2-a2-B-a3-A3-C1-C2.

  FVIII circulates in plasma as two chains that separate at the B-a3 boundary, namely A1-a1-A2-a2-B / a3-A3-C1-C2 heterodimer. The protein structure is stabilized by divalent metal ion binding. The A1-a1-A2-a2-B chain is called the heavy chain (HC), while the a3-A3-C1-C2 chain is called the light chain (LC).

  Endogenous FVIII molecules circulate in vivo as a pool of molecules with B domains of various sizes, the shortest having a C-terminus at position 740, ie, the C-terminus of A2-a2. All such FVIII molecules with different lengths of the B domain have full procoagulant activity. Upon activation by thrombin, FVIII is cleaved at the C-terminus of A1-a1 at position 372, at the C-terminus of A2-a2 at position 740, and between a3 and A3 at position 1689, the latter cleavage being Releases the a3 region and loses its affinity for VWF. The FVIII molecule cleaved (activated) by thrombin is called FVIIIa. Activation allows the interaction of FVIIIa with phospholipid surfaces such as activated platelets and activated factor IX (FIXa), i.e. a tenase complex is formed and factor X (FX ) Can be activated efficiently.

  The terms “Factor VIII (a)” and “FVIII (a)” include both FVIII and FVIIIa. “FVIII (a)” includes naturally occurring allelic variants of FVIII (a) that may exist and occur from one individual to another. FVIII (a) may be obtained from plasma or by recombinant production using well-known production and purification methods. The extent and location of glycosylation, tyrosine sulfation and other post-translational modifications may vary depending on the chosen host cell and its growth conditions.

  Human FVIII consists of 2351 amino acids (including signal peptide) and 2332 amino acids (no signal peptide). The detailed domain structure, A1-a1-A2-a2-B-a3-A3-C1-C2, is represented by the corresponding amino acid residues (see SEQ ID NO: 1): A1 (1-336), a1 (337-372 ), A2 (373-710), a2 (711-740), B (741-1648), a3 (1649-1689), A3 (1690-2020), C1 (2021-2173) and C2 (2174-2332) Have “Native FVIII” is a human FVIII molecule derived from the full-length sequence shown in SEQ ID NO: 1 (amino acids 1 to 2332). The B domain spans amino acids 741-1648 in SEQ ID NO: 1.

SEQ ID NO: 1: wild type human coagulation factor VIII

  The factor VIII molecule contained in the formulation of the present invention may be a B domain truncated / deleted FVIII molecule, in which case the remaining domains are represented by amino acid numbers 1 to 740 and 1649 to 2332 of SEQ ID NO: 1. Corresponds to the sequence to be Thus, these FVIII molecules are preferably recombinant molecules produced in transformed host cells of mammalian origin. However, the remaining domains (i.e., three A-domains, two C-domains and the a1, a2, and a3 regions) have the amino acid sequence represented by SEQ ID NO: 1 (amino acids 1-740 and 1649-2332). May be slightly different, for example, about 1%, 2%, 3%, 4% or 5%.

  The FVIII molecule contained in the preparation of the present invention has a biologically active fragment of FVIII, that is, a domain other than the B domain has been deleted or truncated, but the deleted / truncated FVIII molecule does not form clots. It may be FVIII, which retains its ability to assist. FVIII activity can be assessed in vitro using techniques well known in the art. Examples of FVIII activity assays can be found in the Examples section.

  Factor VIII binding to various other components such as von Willebrand factor (vWF), low density lipoprotein receptor-related protein (LPR), various receptors, other coagulation factors, cell surface, etc. Amino acid modifications (substitutions, deletions, etc.) can be introduced into the remaining domains to alter capacity or to introduce and / or eliminate glycosylation sites. Other mutations that do not abolish FVIII activity can also be imparted to FVIII molecules / analogs for use in the formulations of the present invention.

  As used herein, the term “FVIII analog” refers to one or more amino acids compared to SEQ ID NO: 1 or for a B domain truncated / deleted FVII molecule compared to the corresponding portion of SEQ ID NO: 1. Refers to a FVIII molecule (full length or B domain truncated / deleted) that has been substituted or deleted.

  FVIII analogs are those in which one or more of the amino acid residues of the parent polypeptide are deleted or replaced with other amino acid residues, and / or additional amino acid residues are added to the parent FVIII polypeptide. Including the FVIII molecule.

  In addition, a Factor VIII molecule / analogue can include other modifications, eg, in the truncated B domain and / or in one or more of the other domains of the molecule (“FVIII derivatives”). These other modifications may be made in the form of various molecules conjugated to factor VIII molecules such as, for example, polymeric compounds, peptidic compounds, fatty acid derived compounds, and the like.

  As used herein, the term “FVIII derivative” refers to a sugar PEGylation wherein one or more of the amino acids of the parent FVIII polypeptide is, for example, alkylated, PEGylated [PEG attached to one or more of the glycans of the polypeptide. (e.g., described in US20100261872), acylation, ester formation or amide formation, or conjugating different functional groups such as, for example, a protractive group or a half-life extending moiety to the FVIII polypeptide backbone. Refers to FVIII molecules (full length or B domain truncated / deleted) or FVIII analogs that are chemically modified by other modifications that gate. The term “FVIII derivative” also encompasses a fusion protein in which an FVIII molecule (full length or B domain truncated / deleted) is fused to another polypeptide, such as albumin or an Fc domain or Fc derivative.

  In this context, the term “glyco-pegylated FVIII” refers to a Factor VIII molecule (full-length FVIII) in which one or more PEG groups are attached to the FVIII polypeptide via the polysaccharide side chain (glycan) of the polypeptide. And B domain truncation / deletion type FVIII).

  The term “retarding group” / “half-life extending moiety” as used herein refers to —SH, —OH, —COOH, —CONH 2, —NH 2, or one or more N- and / or O-glycan structures, etc. One or more chemical groups attached to a functional group of one or more amino acid site chains, and when conjugated to a number of therapeutic proteins / peptides, the in vivo circulating half-life of these proteins / peptides It is understood to refer to a chemical group that can be increased.

Examples of retarding groups / half-life extending moieties include biocompatible fatty acids and derivatives thereof, hydroxyalkyl starch (HAS), such as hydroxyethyl starch (HES), poly (Gly _x -Ser _y ) _n [homoamino acid polymer (HAP )], Hyaluronic acid (HA), Heparosan polymer (HEP), polymer based on phosphorylcholine (PC polymer), Fleximer® polymer (Mersana Therapeutics, MA, USA), dextran, poly-sialic acid ( PSA), polyethylene glycol (PEG), Fc domain, transferrin, albumin, elastin-like peptide, XTEN® polymer (Amunix, CA, USA), albumin binding peptide, von Willebrand factor fragment (vWF fragment), Carboxyl-terminal peptide (CTP peptide, Prolor Biotech, IL) and any combination thereof (e.g. McCormick, CL, AB Lowe, and N. Ayres, Wate r-Soluble Polymers, Encyclopedia of Polymer Science and Technology. 2002, John Wiley & Sons, Inc.). The mode of derivatization is not critical and may be as described above.

  The term “Fc fusion protein” is intended herein to include FVIII fused to an Fc domain that may be derived from any antibody isotype. Due to the relatively long circulation half-life of IgG antibodies, IgG Fc domains are often expected to be preferred. For example, the Fc domain may be further modified to modulate certain effector functions, such as complement binding and / or binding to certain Fc receptors. Fusion of an Fc domain with the ability to bind to an FcRn receptor and FVIII will generally increase the circulating half-life of the fusion protein compared to the half-life of wt FVIII.

  Thus, FVIII molecules for use in the present invention may be, for example, FVIII analog fusion proteins, PEGylated or sugar pegylated FVIII analogs or derivatives of FVIII analogs such as FVIII analogs conjugated to heparosan polymers. .

  As used herein, the term “FVIII variant” refers to a group of FVIII analogs and FVIII derivatives.

  Examples of FVIII molecules for use in the formulations of the present invention include, for example, WO2010045568, WO2009062100, WO2010014708, WO2008082669, WO2007126808, US20100173831, US20100173830, US20100168391, US20100113365, US20100113364, WO200331464, WO2009108806, WO2010102886, WO2010115866, PC101 / 051438), WO2011101284 (PCT / EP2011 / 051959), WO2011101277 (PCT / EP2011 / 051889), WO2011131510 (PCT / EP2011 / 055686), WO2012007324 (PCT / EP2011 / 061349), WO2011101267 (PCT / EP2011 / 051723), and Including the FVIII molecule described in WO2013083858.

  Examples of FVIII molecules that can be used in the formulations of the present invention include the active ingredients of Advate®, Helixate®, Kogenate®, Xyntha®, and WO2008 / 135501 and WO2009 / 007451 The FVIII molecule that has been described.

  FVIII molecules contained in the formulations of the present invention are FVIII derivatives that exhibit substantially the same or improved biological activity compared to wild-type FVIII when compared to human FVIII in a chromogenic assay (FVIII activity assay) Alternatively, it can be an FVIII analog or a combination thereof. Examples of FVIII activity assays can be found in the Examples section.

Biological activity The FVIII molecules contained in the formulations according to the present invention function in the coagulation cascade in a manner similar or equivalent to that of human FVIII and interact with FIXa in activated platelets to form FXa. It can guide and help clot formation. FVIII activity can be assessed in vitro using techniques well known in the art. Blood clotting analysis, FX activation assay (often called chromogenic assay), thrombin generation assay and whole blood thromboelastography are examples of such in vitro techniques. FVIII molecules for use in the formulations of the present invention have at least about 10%, at least about 20%, at least about 30%, at least about at least about 10% of the activity of native human FVIII when compared to human FVIII in a chromogenic assay (FVIII activity assay). It may have FVIII activity of greater than about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, 100% or even 100%. Examples of FVIII activity assays can be found in the Examples section.

B domain
The B domain in FVIII spans amino acids 741-1648 of SEQ ID NO: 1. The B domain is cleaved at several different sites, resulting in significant heterogeneity in circulating plasma FVIII molecules. The exact function of the heavily glycosylated B domain is unknown. The B domain is known to be unnecessary for FVIII activity in the coagulation cascade. Thus, recombinant FVIII is often produced in the form of a B domain deleted / truncated molecule. The apparent lack of function is supported by the fact that B domain deleted / truncated FVIII appears to have in vivo properties identical to those found in full-length native FVIII. Nonetheless, there are indications that the B domain may reduce cell membrane binding, at least under serum-free conditions.

B-domain deleted / truncated factor VIII molecule Endogenous full-length FVIII is synthesized as a single-chain precursor molecule. The precursor is cleaved into heavy and light chains before being secreted. Recombinant B domain deleted FVIII can be produced by two different strategies. A heavy chain and a light chain that do not contain a B domain may be synthesized as two different polypeptide chains each (double-chain strategy), or B domain-deleted FVIII as a single precursor polypeptide chain Once synthesized, this may be cleaved into heavy and light chains in the same way as the full length FVIII precursor (single chain strategy).

  In B domain deleted FVIII precursor polypeptides produced by a single chain strategy, the heavy and light chain portions are often separated by a linker. In order to minimize the risk of introducing an immunogenic epitope into B domain deleted FVIII, the linker sequence may be derived from the FVIII B domain. At a minimum, the linker should include a recognition site for a protease that cleaves the B domain deleted FVIII precursor polypeptide into heavy and light chains. In the B domain of full-length FVIII, amino acids 164-1648 constitute this recognition site. The thrombin cleavage site that results in linker removal in the activation of B domain deleted FVIII is located in the heavy chain. Thus, the size and amino acid sequence of the linker is unlikely to affect removal of the linker from the remaining FVIII molecule by thrombin activation. B domain deletion / truncation favors the production of FVIII. Nevertheless, a portion of the B domain can be included in the linker without reducing productivity. The negative effect of B domain on productivity was not due to any particular size or sequence of B domain.

Degradation Factor VIII in liquid formulations is degraded by several mechanisms, including oxidation and dissociation between heavy and light chains. Light chain and heavy chain dissociation can be assessed in vitro by well-known techniques, for example, by size-exclusion chromatography (SEC) as described in the Examples section.

  The dissociation of factor VIII molecules is observed in SEC as the appearance of longer elution time peaks than monomeric factor VIII. This peak was assigned to the free light chain (Fatouros et al., International Journal of Pharmaceutics 155, 121, 1997). Thus, dissociation of FVIII molecules can be assessed, for example, by assaying the level of free light chain.

Embodiments In one embodiment of the invention, the factor VIII is recombinantly produced full length human FVIII. In another embodiment, the factor VIII is recombinantly produced B domain truncated FVIII. In one embodiment of the invention, the factor VIII is full length human FVIII having the sequence shown in SEQ ID NO: 1.

  In one embodiment of the invention, the FVIII molecule is an FVIII sequence analog, and in another embodiment, the FVIII molecule is wild when measured, for example, in a blood clotting assay as described in the experimental section below. An FVIII sequence analog that exhibits substantially the same or improved biological activity compared to type FVIII.

  In one embodiment, the FVIII molecule is a B domain truncated FVIII molecule produced by a vector encoding the amino acid sequence shown in SEQ ID NO: 2, which molecule is a 21 amino acid residue linker sequence (SFSQNSRHPSQNPPVLKRHQR) (SEQ ID NO: Includes 6).

SEQ ID NO: 2:

  In one embodiment, the FVIII molecule is a heavy chain-linker sequence (A1-a1-A2-a2-L) and a light chain sequence (a3-A3-C1-C2) joined together by non-covalent interactions. It is a double-stranded B domain truncated FVIII molecule consisting of The linker (L) is a 20 amino acid residue linker sequence (SFSQNSRHPSQNPPVLKRHQ) (SEQ ID NO: 3), the heavy chain (A1-a1-A2-a2) and the light chain (a3-A3-C1-C2) have the sequence It corresponds to the sequence represented by amino acid numbers 1 to 740 and 1649 to 2332 of number 1, respectively.

  In one embodiment, FVIII is bound to a retarding group. In a series of embodiments, the factor VIII is (i) pegylated FVIII, (ii) FVIII fusion protein, (iii) albumin fusion FVIII or (iv) Fc region fusion FVIII.

  In another embodiment, the factor VIII is a glycopegylated FVIII, such as a full-length human FVIII produced by glycosylated pegylated recombination or a glycopegylated B domain truncated FVIII. In one embodiment, FVII is attached to a polysaccharide (eg, HEP, HES, HAS, PSA). In another embodiment, Factor VIII is bound to polysialic acid (PSA) or heparosan (HEP).

  In one embodiment, the FVIII molecule is a B domain truncated FVIII molecule with a modified circulatory half-life, said molecule shared with a hydrophilic polymer via an O-linked oligosaccharide in the truncated B domain Conjugated by binding, FVIII activation results in the removal of the covalently conjugated polymer. In its different specific embodiments, the hydrophilic polymer is polyethylene glycol (PEG), PSA and HEP.

  In one embodiment, the FVIII molecule is a B domain truncated factor VIII molecule with a modified circulating half-life, said molecule being a hydrophilic polymer via an O-linked oligosaccharide in the cleaved B domain (I) Factor VIII activation results in the removal of the covalently conjugated hydrophilic polymer, and (ii) the heavy and light chain portions of the FVIII precursor polypeptide are , Separated by a linker, the sequence of which is derived from the FVIII B domain. In one embodiment, the length of the B domain is 20-30 amino acids. In one embodiment, the hydrophilic polymer is a polysaccharide, in one embodiment, the polysaccharide is PSA, and in another embodiment, the polysaccharide is HEP. In one embodiment, the hydrophilic polymer is PEG. In different embodiments, the PEG size is from about 10,000 to about 160,000 Da or about 40,000 Da. In a series of embodiments, the PEG used has a size in the range of 2 to 160 kDa or 2 to 80 kDa or 5 to 80 kDa or 5 to 60 kDa or 10 to 80 kDa or 10 to 60 kDa or 20 to 60 kDa. In different embodiments, the PEG is a 2 kDa, 5 kDa, 10 kDa, 20 kDa, 40 kDa or 80 kDa PEG.

  In one particular series of embodiments, the FVIII molecules attached to the above-mentioned retarder group are combined with a heavy chain-linker sequence (A1-a1-A2-a2-L) and a light chain joined together by non-covalent interactions. It is a double-stranded B domain truncated FVIII molecule consisting of a chain sequence (a3-A3-C1-C2), the linker (L) is a 20 amino acid residue linker sequence (SFSQNSRHPSQNPPVLKRHQ) (SEQ ID NO: 3), The chain (A1-a1-A2-a2) and the light chain (a3-A3-C1-C2) correspond to the sequences represented by amino acid numbers 1-740 and 1649-2332 of SEQ ID NO: 1, respectively. In certain embodiments thereof, the FVIII described is (i) one or more PEG groups, (ii) one or more PSA groups, (iii) one or more HEP groups or (iv) It is bound to one or more delay groups selected from PEG, PSA and HEP. In yet another specific embodiment, the one or more delayed PEG / PSA / HEP groups are attached to the FVIII polypeptide via a polysaccharide side chain (glycan) of the polypeptide, yet another embodiment. Wherein the retarding group is attached to the FVIII polypeptide via a glycan located within the linker sequence (SEQ ID NO: 3). In yet another embodiment, the PEG group is 20-60 kDa PEG or 40 kDa PEG.

  In one embodiment, FVIII in the formulations of the present invention are heavy chain-linker sequences (A1-a1-A2-a2-L) and light chain sequences (a3-A3-L) joined together by non-covalent interactions. C1-C2) is a double-stranded B domain truncated FVIII molecule, the linker (L) is a 20 amino acid residue linker sequence (SFSQNSRHPSQNPPVLKRHQ) (SEQ ID NO: 3), heavy chain (A1-a1-A2 -a2) and light chain (a3-A3-C1-C2) correspond to the sequences represented by amino acid numbers 1 to 740 and 1649 to 2332 of SEQ ID NO: 1, respectively, and one or more PEG groups are linkers It is bound to the FVIII polypeptide via a glycan located within the sequence (SEQ ID NO: 3). In yet another embodiment, the PEG group is 20-60 kDa PEG or 40 kDa PEG.

  In one embodiment, FVIII in the formulations of the present invention are heavy chain-linker sequences (A1-a1-A2-a2-L) and light chain sequences (a3-A3-L) joined together by non-covalent interactions. C1-C2) is a double-stranded B domain truncated FVIII molecule, the linker (L) is a 20 amino acid residue linker sequence (SFSQNSRHPSQNPPVLKRHQ) (SEQ ID NO: 3), heavy chain (A1-a1-A2 -a2) and light chain (a3-A3-C1-C2) correspond to the sequences represented by amino acid numbers 1 to 740 and 1649 to 2332 of SEQ ID NO: 1, respectively, and one or more HEP groups are linkers It is bound to the FVIII polypeptide via a glycan located within the sequence (SEQ ID NO: 3).

  In one embodiment, FVIII in the formulations of the present invention are heavy chain-linker sequences (A1-a1-A2-a2-L) and light chain sequences (a3-A3-L) joined together by non-covalent interactions. C1-C2) is a double-stranded B domain truncated FVIII molecule, the linker (L) is a 20 amino acid residue linker sequence (SFSQNSRHPSQNPPVLKRHQ) (SEQ ID NO: 3), heavy chain (A1-a1-A2 -a2) and light chain (a3-A3-C1-C2) correspond to the sequences represented by amino acid numbers 1 to 740 and 1649 to 2332 of SEQ ID NO: 1, respectively, and one or more PSA groups are linkers It is bound to the FVIII polypeptide via a glycan located within the sequence (SEQ ID NO: 3).

  In one embodiment, the FVIII in the formulation of the invention is a B domain truncated FVIII molecule as shown in SEQ ID NO: 2 and the one or more PEG groups are via a glycan located within the linker sequence (SEQ ID NO: 3). Bound to the FVIII polypeptide. In yet another embodiment, the PEG group is 20-60 kDa PEG or 40 kDa PEG.

  In one embodiment, FVIII in a formulation of the invention is a B domain truncated FVIII molecule as shown in SEQ ID NO: 2 and the one or more HEP groups are via a glycan located within the linker sequence (SEQ ID NO: 3). Bound to the FVIII polypeptide.

  In one embodiment, FVIII in the formulation of the invention is a B domain truncated FVIII molecule as shown in SEQ ID NO: 2 and the one or more PSA groups are via a glycan located within the linker sequence (SEQ ID NO: 3). Bound to the FVIII polypeptide.

Aqueous Formulation The concentration of Factor VIII in the formulations of the present invention is typically in the range of 10 to 10,000 IU / mL. In different embodiments, the concentration of FVIII molecules in the formulations of the invention is 10-8000 IU / mL, or 10-6000 IU / mL, or 10-4000 IU / mL, or 10-2500 IU / mL, or 30-4000 IU / mL, Or in the range of 30-2500 IU / mL, or 50-2500 IU / mL, or 50-1250 IU / mL, or 100-2500 IU / mL.

  1 IU (international units) is defined as the amount of FVIII present in 1 mL of fresh, pooled normal human plasma.

  In one embodiment of the invention, the pharmaceutical formulation is an aqueous solution. The term “aqueous formulation” is defined as a formulation comprising at least 50% by weight of water. Similarly, the term “aqueous solution” is defined as a solution comprising at least 50% by weight of water.

Salt The preparation according to the present invention comprises a calcium salt. The formulation can also contain a sodium salt.

  In one embodiment, the formulation contains at least 15 mM calcium salt. In one series of embodiments, the formulation is 15-100 mM calcium salt, or 15-80 mM, or 15-60 mM, or 15-45 mM, or 15-30 mM, or 15-25 mM, or 15-20 mM, or at least 20 mM. Calcium salt, or 20-100 mM, or 20-80 mM, or 20-60 mM, or 20-45 mM, or 20-45 mM, or 20-40 mM, or 20-30 mM, or 25-35 mM, or at least 30 mM, or 30 Contains ~ 45 mM, or about 30 mM. In one particular embodiment, the fomulation contains 25-35 mM calcium salt.

  The calcium salt can be selected, for example, from the group of calcium chloride, calcium acetate, calcium lactate, calcium benzoate and mixtures thereof, and other soluble calcium salts well known to those skilled in the art. In one embodiment, the calcium salt is calcium chloride.

  In one embodiment, the formulation does not contain EDTA.

  In one embodiment, the formulation contains at least 5 mM sodium salt. In one series of embodiments, the formulation is 5 to 500 mM sodium salt, or 15 to 150 mM, or 15 to 125 mM, or 15 to 100 mM, or at least 20 mM calcium salt, or 20 to 150 mM, or 20 -130 mM, or 20-100 mM, or at least 30 mM, or 30-150 mM, or 50-150 mM. In one embodiment, the concentration of sodium salt is 100 mM or 5-100 mM or 50-100 mM or less than 50 mM or 5-50 mM.

  The sodium salt used for pH adjustment is typically in the form of NaOH and is included at a defined sodium concentration.

  The sodium salt can be selected, for example, from the group of sodium chloride, sodium acetate, sodium lactate, sodium benzoate and mixtures thereof, and other soluble sodium salts well known to those skilled in the art.

  In one embodiment of the invention, the sodium salt is sodium chloride, and in another embodiment, the salt is sodium acetate. In a third embodiment, the formulations of the present invention contain a mixture of sodium chloride and sodium acetate.

Buffer The formulation according to the present invention may comprise a buffer system. Buffers (or buffer substances) include acetate, benzoate, carbonate, citrate, glycylglycine, histidine or histidine derivatives, Hepes, glycine, phosphate, hydrogen phosphate and tris (hydroxymethyl). -May be selected from the group consisting of aminomethane (TRIS), bicine, tricine, succinate, aspartic acid, glutamic acid or mixtures thereof. In one embodiment of the present invention, the concentration of the buffer substance is 1-100 mM, such as 1-50 mM, 1-25 mM, 1-20 mM, 5-20 mM, 5-15 mM, or the like.

  In one embodiment of the invention, the formulation comprises histidine, preferably L-histidine. In one embodiment thereof, the concentration of histidine is 1-100 mM, such as 1-50 mM or 1-25 mM or 1-20 mM or 5-20 mM or 5-15 mM.

  The liquid formulation of the present invention typically has a pH of 5.5 to 7.5. In different embodiments, the formulation has a pH of 6.0-7.0 or 6.2-6.8 or 6.3-6.7.

Sugar and / or Polyol The preparation of the present invention further comprises a sugar (sugar) and / or a polyol (sugar alcohol). The saccharide is selected, for example, from the group of monosaccharides, disaccharides or polysaccharides and water-soluble glucans (including monosaccharide fructose, glucose, mannose, disaccharide lactose, sucrose, trehalose and polysaccharides dextran, raffinose, stachyose) Can be done. Polyols include, for example, sugar alcohols (including mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol), alditols (for example, glycerol (glycerin), 1,2-propanediol (propylene glycol), 1, 3-propanediol, 1,3-butanediol], polyethylene glycol, or a mixture thereof.

  If more than one saccharide and / or polyol is included in the formulation, the stated concentration shall indicate the total amount of “saccharide and / or polyol” present in the formulation.

  In one embodiment of the invention, the FVIII formulation comprises one or more saccharides and / or sugar alcohols selected from the group of sucrose, sorbitol, glycerol, raffinose, stachyose, mannitol, sorbitol, or mixtures thereof.

  In one embodiment, the FVIII formulation comprises a saccharide and / or sugar alcohol at a concentration of at least 200 mM.

  In one embodiment, the FVIII formulation according to the invention comprises one or more saccharides and no polyols. In another embodiment, the formulation comprises a single saccharide component and no polyol. In the specific embodiment described above, the saccharide is sucrose.

  In one embodiment, the formulation comprises one or more polyols and no saccharides. In another embodiment, the formulation comprises a single polyol component and no sugars. In the specific embodiments described above, the saccharide is sorbitol or mannitol.

In one embodiment of the invention, the formulation comprises a concentration of sugar and / or sugar alcohol that results in a calculated osmotic concentration (“X”) of the formulation of 1.50 Osm / L or less. Thus, in one embodiment of the invention, the formulation comprises a saccharide and / or sugar alcohol at a concentration between 100 mM and XmM, wherein X is a value (mM) at which the calculated osmotic concentration of the formulation reaches 1.50 Osm / L. Is defined as In another embodiment, the formulation comprises a saccharide and / or sugar alcohol at a concentration between 200 mM and XmM, where X is defined as the value (mM) at which the calculated osmolarity of the formulation reaches 1.50 Osm / L. The In determining the X value of the formulation (Fomulation) problem by calculating the osmolality of a given formulation, into account all the components of the formulation (e.g., CaCl _2, NaCl, buffer substances, methionine) . The calculation of osmotic concentration (concetration) is described in this application (see the “Osmotic Concentration” section below). However, the theoretical calculation of osmotic concentration is well known to those skilled in the art.

  In one embodiment of the invention, the formulation is at least 100 mM, or at least 200 mM, or 100-1800 mM, or 300-1800 mM, or 100-1500 mM, or 200-1800 mM, or 200-1500 mM, or 100-1000 mM, or 200. Containing sugars and / or sugar alcohols at a concentration of ~ 1000 mM, or 300-1000 mM, or 200-800 mM, or 300-800 mM, or 400-800 mM, or 500-800 mM, or 500-700 mM.

  In one embodiment, the formulation of the present invention comprises sucrose. In one embodiment, the concentration of sucrose is 100-1000 mM, or 150-1750 mM, or 200-1000 mM, or 300-1000 mM, or 200-800 mM, or 300-800 mM, or 440-730 mM, or 400-800 mM, or In another embodiment, the concentration of sucrose is 50-600 mg / mL, or 100-600 mg / mL, or 100-450 mg / mL, or 150-450 mg / mL, Or 150-250 mg / mL (100 mg / mL sucrose corresponds to 292 mM).

  In another embodiment, the formulations of the present invention include sorbitol. In different embodiments, the concentration of sorbitol is at least 400 mM, or 400-1500 mM, or 100-800 mg / mL, or 100-650 mg / mL, or 150-650 mg / mL, or 150-500 mg / mL, or 150-250 mg. (100 mg / mL sorbitol corresponds to 549 mM).

Other excipients The formulations of the present invention may further contain additional excipients. Examples of standard excipients for use in the pharmaceutical formulations according to the invention are preservatives, antioxidants and surfactants.

  In one embodiment of the invention, a reducing agent such as methionine (or other sulfated amino acid or sulfated amino acid analog) can be added to inhibit oxidation of methionine residues to methionine sulfoxide. “Inhibition” intends to minimize the accumulation of methionine oxidized species during production and over time. Inhibition of methionine oxidation results in better retention of the polypeptide in its proper molecular form. The amount to be added should be sufficient to inhibit the oxidation of methionine residues so that the amount of methionine sulfoxide allowed by the regulatory authority is reached. Typically this means that the formulation contains from about 10% to about 30% or less methionine sulfoxide. In general, this can be achieved by adding methionine such that the ratio of methionine added to the methionine residue of Factor VIII is at least about 1: 1.

  In one embodiment of the invention, the formulation comprises methionine, such as L-methionine. In one embodiment thereof, the concentration of methionine is 0.05-100 mM, such as 0.1-10 mM or 0.1-2 mM or 0.2-0.5 mM.

  In one embodiment of the invention, the formulation further comprises a surfactant. Typical surfactants (trademark examples are shown in square brackets []) are polyoxyethylene (20) sorbitan monolaurate [Tween 20], polyoxyethylene (20) sorbitan monopalmitate [Tween 40]. Or polyoxyethylene sorbitan fatty acid ester such as polyoxyethylene (20) sorbitan monooleate [Tween 80], polyoxypropylene-polyoxyethylene block copolymer [Pluronic F68 / poloxamer 188], polyethylene glycol octylphenyl ether [ Poloxamers such as Triton X-100] or polyoxyethylene glycol dodecyl ether [Brij 35]. The use of surfactants in pharmaceutical formulations is well known to those skilled in the art. For convenience, see Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

  In one embodiment of the invention, the formulation comprises sorbitan monooleate [Tween 80]. In one embodiment thereof, the concentration of sorbitan monooleate [Tween 80] is 0.01-0.5 mg / mL, such as 0.05-0.3 mg / mL or 0.05-0.2 mg / mL or about 0.1 mg / mL. is there.

  In one embodiment of the invention, the formulation comprises poloxamer 188. In one embodiment thereof, the concentration of poloxamer 188 is 0.01-5 mg / mL, such as 0.05-3 mg / mL or 0.25-2 mg / mL or about 0.5 mg / mL.

In one embodiment of the invention, the formulation contains FVIII, L-histidine, Tween® 80, L-methionine, NaCl, sucrose and CaCl ₂ , and in another embodiment of the invention, the formulation is FVIII, 10 mM L-histidine, 0.1 mg / ml Tween® 80, 0.37 mM L-methionine, 78 mM NaCl, 188 mg / ml sucrose and 30 mM CaCl ₂ , pH 6.7, in another embodiment , the formulation comprises FVIII, 20 to 40 mM CaCl _2, the 500~800mM sucrose or 150 to 250 / mL sorbitol. In certain embodiments of any of these embodiments, the FVIII is
(i) an FVIII molecule comprising a domain corresponding to the sequence represented by amino acid numbers 1 to 740 and 1649 to 2332 of SEQ ID NO: 1, or
(ii) the B domain truncated FVIII molecule shown in SEQ ID NO: 2, or
(ii) Double chain B consisting of heavy chain-linker sequence (A1-a1-A2-a2-L) and light chain sequence (a3-A3-C1-C2) joined together by non-covalent interactions A domain-cut FVIII molecule, wherein the linker (L) is a 20 amino acid residue linker sequence (SFSQNSRHPSQNPPVLKRHQ) (SEQ ID NO: 3), a heavy chain (A1-a1-A2-a2) and a light chain (a3- A3-C1-C2) is a molecule corresponding to the sequence represented by amino acid numbers 1 to 740 and 1649 to 2332 of SEQ ID NO: 1, respectively, or
(iv) B domain truncated FVIII molecule shown in SEQ ID NO: 2, wherein one or more PEG groups are linked to the FVIII polypeptide via a glycan located within the linker sequence (SEQ ID NO: 3) Molecule, or
(v) Double chain B consisting of heavy chain-linker sequence (A1-a1-A2-a2-L) and light chain sequence (a3-A3-C1-C2) joined together by non-covalent interactions A domain-cut FVIII molecule, wherein the linker (L) is a 20 amino acid residue linker sequence (SFSQNSRHPSQNPPVLKRHQ) (SEQ ID NO: 3), a heavy chain (A1-a1-A2-a2) and a light chain (a3- A3-C1-C2) corresponds to the sequences represented by amino acid numbers 1 to 740 and 1649 to 2332 of SEQ ID NO: 1, respectively, and one or more PEG groups are located within the linker sequence (SEQ ID NO: 3) A molecule that binds to an FVIII polypeptide via a glycan that
(vi) a B domain truncated FVIII molecule having a modified circulatory half-life, wherein the molecule is covalently conjugated to a hydrophilic polymer via an O-linked oligosaccharide in the cleaved B domain. FVIII activation results in the removal of the covalently conjugated polymer, and in its different specific embodiments, the molecules wherein the hydrophilic polymer is polyethylene glycol (PEG), PSA and HEP,
(vii) a B domain truncated factor VIII molecule having a modified circulatory half-life, wherein the molecule is covalently conjugated to a hydrophilic polymer via an O-linked oligosaccharide in the cleaved B domain. (I) Factor VIII activation results in the removal of the covalently conjugated hydrophilic polymer, and (ii) the heavy and light chain portions of the FVIII precursor polypeptide are separated by a linker. A molecule wherein the sequence of the linker is derived from the FVIII B domain, and in one embodiment the length of the B domain is 20-30 amino acids, or
(vi) Active® active ingredient, or
(vii) Helixate® active ingredient, or
(viii) an active ingredient of Kogenate®, or
(ix) Xyntha® active ingredient, or
(x) a FVIII molecule produced as described in Thim L. et al. (Haemophilia 2010; 16: 349-359), or
(xi) FVIII molecule produced as described in WO2009108806.

Antioxidant Antioxidant effects can be achieved by replacing oxygen (air) so that it does not come into contact with the product. In certain embodiments, the formulation does not contain an antioxidant, and instead the sensitivity of FVIII to oxidation is by eliminating the atmosphere or replacing oxygen (air) so that it does not come into contact with the product. Be controlled. This can be achieved, for example, by saturating the liquid formulation with either nitrogen, helium or argon and sealing the final container after replacing the air on the product with the gas. The operation of replacing oxygen (air) may be performed, for example, as a `` degassing '' process, in which case (i) exposure to an inert gas (argon, helium or nitrogen) and / or (ii) the formulation The formulation is processed by performing one or more cycles of evacuating the containing chamber to a pressure below atmospheric pressure. In one particular embodiment, the formulation is sterile filtered, dispensed into vials, degassed by 3 cycles of pure N ₂ exposure, interspersed by briefly evacuating the chamber to 0.1 bar pressure, then the head Fill the space with pure N ₂ and seal the vial.

  Of course, the use of antioxidants may be combined with atmospheric exclusion. Further, the formulation may be protected from light, and of course the protection may be combined with either or both atmospheric exclusion and the use of antioxidants.

  Thus, the present invention also provides an airtight container [eg, vial or cartridge (such as a cartridge for a pen-type applicator)] containing a liquid aqueous pharmaceutical formulation as defined herein and optionally an inert gas. I will provide a. The inert gas can be selected from the group consisting of nitrogen, helium or argon. In this context, the term “airtight container” means a container that has a low permeability to oxygen (air). Containers (e.g. vials or cartridges or syringes) are typically glass or plastic closed by rubber closures or other closure means that allow penetration while preserving the integrity of the pharmaceutical formulation as needed , Especially made of glass. In yet another embodiment, the container is a vial or cartridge enclosed in a sealed bag, eg, a sealed plastic bag such as a laminated type [eg, a metal (aluminum, etc.) laminated plastic bag].

Administration and Treatment In one embodiment of the present invention, the formulation is a pharmaceutical formulation contemplated for administration to a subject. The formulation is typically administered by parenteral administration, which can be done by syringe, optionally subcutaneous, intramuscular, intraperitoneal or intravenous injection using a pen-like syringe. . Alternatively, parenteral administration can be performed using an infusion pump.

  The present invention also encompasses a method for treating hemophilia A, comprising the step of administering a formulation according to the present invention to a subject in need thereof.

  As used herein, the term “subject” includes any human patient or non-human vertebrate.

  As used herein, the term “treating” or “treatment” refers to a medical therapy for any human or other vertebrate subject in need thereof. The subject has undergone a physical examination by a doctor or veterinarian, and the doctor or veterinarian indicates that the use of the specific treatment is beneficial to the health of the human or other vertebrate animal It is expected that The timing and purpose of the treatment can vary from individual to individual depending on the health status of the subject. Thus, the treatment can be a prophylactic, palliative, symptomatic and / or curative treatment. In view of the present invention, prophylactic, palliative, symptomatic and / or curative treatments can represent separate embodiments of the present invention.

  Said hemophilia A may be severe, moderate or mild. The clinical severity of hemophilia A is determined by the concentration of FVIII functional units in the blood and is classified as mild, moderate or severe. Severe hemophilia is defined by <0.01 U / ml clotting factor levels, which corresponds to <1% of normal levels, while moderate and mild patients have 1-5% and> 5% levels, respectively .

Osmotic concentration Osmotic concentration, formerly known as osmolarity, is a measure of solute concentration, defined as the number of osmoles of solute per liter (L) of solution (Osm) (osmol / L or Osm / L). It is. Volumetric molarity measures the number of moles of solute per unit volume of solution, while osmolarity measures the number of osmoles of solute particles per unit volume of solution. Osmolality is a measure of the solute's osmolality per kilogram of solvent (osmol / kg or Osm / kg). The molarity and osmolarity are theoretically temperature dependent. This occurs because water changes its capacity with temperature. However, when the solute concentration is low, the osmolarity and osmolality are considered equivalent.

  Theoretical calculations of osmotic pressure concentrations are well known to those skilled in the art. Briefly, for each solution component, the product of the osmotic pressure f, the number of particles n dissociating in water and the molar concentration is calculated and the results for all components are summed.

Thus, the osmotic concentration of the solution can be calculated from the following formula: Osm / L = Σ _i f _i n _i C _i (where the index i represents the identity of a particular component; f _i Is the osmotic pressure coefficient of a particular component; n is the number of particles from which the molecule dissociates in water; C is the molar concentration of the component). As stated earlier, the molarity has a slight temperature dependence; for the purposes of the present invention we refer to the concentration at 25 ° C.

  An alternative method for assessing the osmotic pressure that can be generated by the solution after injection is by osmolality assessment, in which the content of the component is assessed relative to the solvent mass. If the density of the solution and the dry mass of the dissolved component are known, the osmolality and osmotic concentration can be easily interconverted. Osmolality can be measured in a number of ways, most commonly by freezing point depression. For example, with respect to water, 1 osmol of solute added to 1 kg of water lowers the freezing point by 1.86 ° C. Methods for determining the osmolality of a solution by freezing point depression are described, for example, in European Pharmacopoeia 2.2.35 and US Pharmacopeia Chapter 785.

  The table below lists the osmotic coefficient and particle number n of some important excipients. For other components, a value of f = 1 gives a good estimate sufficient for practical purposes, the value of n being well known for virtually all compounds associated with pharmaceutical preparations for parenteral use It is.

  In different embodiments of the invention, the formulation has a calculated osmotic concentration below 1.50 Osm / L, below 1.20 Osm / L, below 1.00 Osm / L or below 0.90 Osm / L.

Accelerated assay for confirming the stabilizing effect of excipients in FVIII preparations Therefore, the present inventors have developed a liquid preparation of a multivalent protein by an accelerated assay in which a sample including a chemical protein denaturant is incubated in a short time. It was further discovered that the stabilizing effect of the excipient in can be accurately confirmed.

  Thus, incubation of protein denaturants with liquid formulations of factor VIII molecules (including analogs and derivatives) followed by subsequent analysis [eg by molecular sieve chromatography (SEC) as described in the Examples section ] Is a useful tool for rapid drug product investigation. Samples can also be analyzed for activity, for example, by the chromogenic assay described in the Examples section. The method according to the present invention provides a quick and reliable method of performing accelerated stability testing. The method provides a method of avoiding long term storage of the sample and / or avoiding subjecting the test sample to stress conditions. Stress conditions can make it difficult to extrapolate the obtained results under real-time conditions. The method according to the present invention provides a method for quickly and accurately identifying a sample of an FVIII formulation with low free light chain formation, i.e. a method for quickly and accurately identifying a stable formulation.

  If it takes months or even years to obtain real-time stability data for a given formulation, the method can be accomplished in a short period of time, typically within a week, typically 24 to 48 hours. Provide data within.

  Chemical protein denaturants are characterized by destabilizing the protein structure by the composition of the solution rather than by external stresses such as extreme temperature, mechanical stress or light. Examples of chemical protein denaturing agents are chaotropic agents such as guanidinium chloride, urea, thiourea, ethanol and other compounds well known to those skilled in the art. Conditions with a pH below 5.5 or above 8.0 may also function as a chemical modifier of Factor VIII.

  In one embodiment of the invention, the modifier is guanidinium chloride (GuHCl). In another embodiment, the denaturing agent is urea. In one series of embodiments, the denaturing agent is guanidinium chloride at a concentration such as 0.1 to 1.0M, such as 0.2 to 0.8M or 0.2M or 0.4M. In another series of embodiments, the denaturing agent is urea at a concentration such as 1-5M, such as 1-3M or 1-2M.

  The formulation can be analyzed by any method that searches for the presence of intact factor VIII molecules. Particularly suitable are chromatographic methods that provide separate signals for intact factor VIII molecules and either dissociated light or dissociated heavy chains, or separate signals for all three species.

  In one embodiment, the formulation is analyzed by molecular sieve chromatography. In another embodiment, the formulation is analyzed by field flow fractionation. In another embodiment, the formulation is analyzed by ion exchange chromatography. In another embodiment, the formulation is analyzed by hydrophobic interaction chromatography. In another embodiment, the formulation is analyzed by analytical ultracentrifugation. Other separation methods well known to those skilled in the art can be used as well.

Incubation time and temperature:
After the denaturant is added to the FVIII formulation, the denaturant-containing formulation is typically incubated at about 5 ° C. for at least 1 hour, more preferably 24 hours or more. In different embodiments, the incubation time is 12-240 hours, 12-120 hours, or 24-120 hours, or 24-60 hours.

List of Embodiments A number of different embodiments of the invention will now be described.

Embodiment 1: A liquid aqueous formulation of coagulation factor VIII comprising a factor VIII polypeptide, a calcium salt at a concentration of at least 15 mM, and a sodium salt at a concentration of at least 10 mM and having a pH of 6.0 to 7.5.

Embodiment 2: The formulation of embodiment 1, comprising a calcium salt at a concentration of 20-45 mM.

Embodiment 3: The formulation of embodiment 1 or embodiment 2, wherein the salt is calcium chloride.

Embodiment 4: The formulation according to any one of Embodiments 1-3, wherein the concentration of sodium salt is at most 100 mM.

Embodiment 5: The formulation according to any one of the previous embodiments, wherein the sodium salt is sodium chloride or sodium acetate.

Embodiment 6: The formulation of any one of Embodiments 1-5, further comprising a saccharide or sugar alcohol at a concentration of at least 200 mM.

Embodiment 7: The formulation of embodiment 6, comprising sucrose at a concentration of at least 200 mM and at most 800 mM.

Embodiment 8: The formulation of embodiment 6, comprising sorbitol at a concentration of at least 400 mM.

Embodiment 9: The formulation of any one of Embodiments 1-8, wherein the Factor VIII polypeptide is recombinant full length FVIII or recombinant B domain truncated FVIII.

Embodiment 10: A formulation according to embodiment 9, wherein the factor VIII polypeptide is conjugated to a retarding group.

Embodiment 11: A formulation according to embodiment 10, wherein the Factor VIII polypeptide is pegylated FVIII or albumin fusion FVIII or Fc region fusion FVIII.

Embodiment 12: A formulation according to embodiment 10, wherein the Factor VIII polypeptide is glycopegylated B domain truncated FVIII.

Embodiment 13: The formulation according to any one of the preceding embodiments, having a pH of 6.0 to 7.0 or 6.4 to 7.0.

Embodiment 14: A method for optimizing a liquid formulation of coagulation factor VIII comprising:
(i) preparing various liquid formulations containing Factor VIII to be tested;
(ii) adding a protein denaturant to the liquid formulation and incubating the resulting solution for a predetermined period;
(iii) analyzing the incubated solution of (ii) for the presence of free FVIII light chain;
(iv) selecting one or more formulations having the desired low level of free light chain.

Embodiment 15: A method for identifying a stable liquid formulation of Factor VIII comprising:
(i) preparing various liquid formulations containing Factor VIII to be tested;
(ii) adding a protein denaturant to the liquid formulation and incubating the resulting solution for a predetermined period;
(iii) analyzing the incubated solution of (ii) for the presence of free FVIII light chain;
(iv) selecting one or more formulations having the desired low level of free light chain.

Embodiment 16: A method according to embodiment 14 or embodiment 15, wherein the protein denaturant is guadinium chloride or urea.

Embodiment 17: A method according to any one of embodiments 14 to 16, wherein the factor VIII polypeptide is recombinant full length FVIII or recombinant B domain truncated FVIII.

Embodiment 18: A method according to embodiment 17, wherein the Factor VIII polypeptide is attached to a retarding group.

Embodiment 19: A method according to embodiment 18, wherein the Factor VIII polypeptide is pegylated FVIII or albumin fusion FVIII or Fc region fusion FVIII.

Embodiment 20: A method according to embodiment 19, wherein the factor VIII polypeptide is glycopegylated B-domain truncated FVIII.

Still another embodiment will be described below.
Embodiment 21. A liquid aqueous formulation of coagulation factor VIII comprising a Factor VIII polypeptide, a calcium salt at a concentration of at least 15 mM, and a polyol at a concentration of at least 100 mM and having a pH of 5.5-7.0.

Embodiment 22: The formulation of embodiment 21, comprising a calcium salt at a concentration of 15-100 mM or 20-45 mM.

Embodiment 23: The formulation according to embodiment 21 or embodiment 22, wherein the salt is calcium chloride.

Embodiment 24: A formulation according to any one of the embodiments 21 to 23, wherein the calculated osmotic concentration is at most 1500 mOsm / L or 1000 mOsm / L or 900 mOsm / L.

Embodiment 25: The formulation according to any one of the embodiments 21-24, further comprising a sodium salt.

Embodiment 26: The formulation according to embodiment 25, wherein the sodium salt is sodium chloride or sodium acetate, or a mixture thereof.

Embodiment 27: The formulation according to any one of embodiments 21 to 26, wherein the polyol is a saccharide or a sugar alcohol.

Embodiment 28: The formulation of embodiment 27, comprising sucrose at a concentration of at least 200 mM and at most 800 mM.

Embodiment 29: A formulation according to embodiment 27, comprising sorbitol at a concentration of at least 400 mM.

Embodiment 30: The formulation according to any one of embodiments 21 to 29, wherein the factor VIII polypeptide is recombinant full length FVIII or recombinant B domain truncated FVIII.

Embodiment 31 A formulation according to any one of embodiments 21 to 30, wherein the Factor VIII polypeptide is conjugated to a retarding group.

Embodiment 32: A formulation according to embodiment 31, wherein the Factor VIII polypeptide is pegylated FVIII or albumin fusion FVIII or Fc region fusion FVIII.

Embodiment 33: A formulation according to embodiment 31 or embodiment 32, wherein the factor VIII polypeptide is glycopegylated B domain truncated FVIII.

Embodiment 34 The formulation according to any one of the embodiments 21 to 33, which has a pH of 6.0 to 7.0 or 6.4 to 7.0.

Embodiment 35: A method for optimizing a liquid formulation of coagulation factor VIII comprising:
(i) preparing various liquid formulations containing Factor VIII to be tested;
(ii) adding a protein denaturant to the liquid formulation and incubating the resulting solution for a predetermined period;
(iii) analyzing the incubated solution of (ii) for the presence of free FVIII light chain;
(iv) selecting one or more formulations having the desired low level of free light chain.

Embodiment 36: A method for identifying a stable liquid formulation of Factor VIII comprising:
(i) preparing various liquid formulations containing Factor VIII to be tested;
(ii) adding a protein denaturant to the liquid formulation and incubating the resulting solution for a predetermined period;
(iii) analyzing the incubated solution of (ii) for the presence of free FVIII light chain;
(iv) selecting one or more formulations having the desired low level of free light chain.

Embodiment 37: A method according to embodiment 35 or embodiment 36, wherein the protein denaturant is guanidinium chloride or urea.

Embodiment 38: A method according to any one of embodiments 35 to 37, wherein the Factor VIII polypeptide is recombinant full length FVIII or recombinant B domain truncated FVIII.

Embodiment 39: A method according to embodiment 38, wherein the Factor VIII polypeptide is attached to a retarding group.

Embodiment 40: A method according to embodiment 39, wherein the Factor VIII polypeptide is pegylated FVIII or albumin fusion FVIII or Fc region fusion FVIII.

Embodiment 41: A method according to embodiment 39 or embodiment 40, wherein the factor VIII polypeptide is glycopegylated B domain truncated FVIII.

List of experimental abbreviations
SEC molecular sieve chromatography
LC light chain
GuHCl guanidinium chloride
BDD-FVIII B domain deleted / truncated factor VIII
GP-BDD-FVIII Glycopegylated B domain truncation / deletion factor VIII

Production of recombinant B domain truncated / deleted FVIII
B domain truncated / deleted FVIII (`` BDD-FVIII '') (SEQ ID NO: 2):
The production of BDD-FVIII is described, for example, in Thim L. et al. (Haemophilia 2010; 16: 349-359).

GlycoPEGylated B domain truncated / deleted FVIII (`` GP-BDD-FVIII ''):
The production of GP-BDD-FVIII is described, for example, in WO2009 / 108806.

FVIII-Fc / albumin fusion protein:
Fusion protein in which factor VIII is fused to Fc domain (SEQ ID NO: 4) or albumin (SEQ ID NO: 5), respectively, by transient expression in HEK cells followed by three-step purification on affinity columns F25 Sepharose and Poros 50 HQ Was prepared.

SEQ ID NO: 4-Fc fusion:

SEQ ID NO: 5-albumin fusion:

FVIIIa activity assay: Chromogenic assay
The FVIII activity (FVIII: C) of rFVIII compounds is evaluated in a chromogenic FVIII assay using Coatest® SP FVIII reagent (Chromogenix) as follows: rFVIII samples and FVIII standards (e.g. from NIBSC) Dilute purified wild-type rFVIII, calibrated against the 7th international FVIII standard, with Coatest® assay buffer (50 mM Tris, 150 mM NaCl, 1% BSA, pH 7.3, preservative included) To do. 50 μl of sample, standard and buffer negative control are added in duplicate to a 96 well microtiter plate (Nunc). Coatest® SP kit Factor IXa / Factor X reagent, phospholipid reagent and CaCl ₂ are mixed 5: 1: 3 (vol: vol: vol) and 75 μl is added to the wells. After 15 minutes incubation at room temperature, 50 μl of factor Xa substrate S-2765 / thrombin inhibitor I-2581 mix is added and the reagents are incubated for 10 minutes at room temperature, followed by 25 μl of 1M citric acid, pH 3. The absorbance at 415 nm is measured in a Spectramax® microtiter plate reader (Molecular Devices) using the absorbance at 620 nm as the reference wavelength. A negative control value is subtracted from all samples and a calibration curve is generated by linear regression of the absorbance values plotted against FVIII concentration.

FVIIIa activity assay: One-stage blood coagulation assay
The FVIII activity (FVIII: C) of rFVIII compounds is further evaluated in a one-step FVIII blood coagulation assay as follows: rFVIII samples and FVIII standards (e.g. calibrated against the 7th International FVIII standard from NIBSC) , Purified wild type rFVIII) is diluted with HBS / BSA buffer (20 mM hepes, 150 mM NaCl, pH 7.4, containing 1% BSA) to approximately 10 U / ml, followed by FVIII deficient plasma containing VWF ( Dade Behring)). The sample is then diluted in HBS / BSA buffer. APTT blood clotting time is measured using an ACL300R or ACL5000 instrument (Instrumentation Laboratory) using a single factor program. VWF-containing FVIII deficient plasma (Dade Behring) is used as assay plasma and SynthASil® (Hemosil®, Instrumentation Laboratory) is used as PTT reagent. In a blood clotting apparatus, the diluted sample or standard is mixed with FVIII deficient plasma and PTT reagent at 37 ^° C. The time until the clot is formed is determined by adding calcium chloride and measuring the turbidity. The FVIII: C in the sample is calculated based on a standard curve of clot formation time for dilutions of FVIII standard.

FVIII degradation: Determination of FVIII free light chain by molecular sieve chromatography (SEC) The dissociation of the rFVIII compound to produce free heavy and light chains is assessed by the SEC method. The column is Sepax Zenix ™ SEC-300 and the eluent is 10 mM Tris, 10 mM CaCl ₂ , 300 mM NaCl and 5% isopropanol, pH 7.0. Factor VIII molecule degradation is observed in SEC as the appearance of peaks with longer elution times than monomeric factor VIII. This peak was assigned to the free light chain (free LC).
Practical example (WORKING EXAMPLES)

In all formulations, a series of formulations of glycopegylated factor VIII (GP-BDD-FVIII) was prepared containing the following components: 28 μg / mL glycopegylated factor VIII, 18 mg / mL NaCl, 0.1 mg / mL mL polysorbate 80,0.6Mg / mL sucrose, 0.055 mg / mL methionine, 1.5 mg / mL _{histidine, 0.13mg / mL CaCl 2, pH6.5} . In addition, each formulation contained additional polyol stabilizers as listed in the following table 1 (Table 2).

  Samples were incubated for 5 weeks at 5 ° C. and analyzed for free light chain by SEC (described above). In addition, a sample set with the same formulation but also containing 0.2M GuHCl was prepared and incubated for 24 hours at 5 ° C., followed by analysis for free light chain by SEC. The relative areas of the free light chain peaks in the two experiments are listed in the following table 2 (Table 3).

  It can be seen that the rapid method with a chemical denaturant accurately confirms that formulation 3 had minimal free light chain formation at 5 ° C. for 5 weeks.

  To investigate the optimal calcium concentration in a liquid formulation of glycopegylated factor VIII (GP-BDD-FVIII), approximately 250 U / mL glycopegylated factor VIII, 18 mg / mL NaCl, 0.05 mg / mL polysorbate 80, 1.5 A formulation with mg / mL sucrose, 1 mg / mL methionine and 1.5 mg / mL histidine pH 6.9 was prepared. The calcium chloride concentrations are listed in the following table 3 (Table 4).

The solution is sterile filtered, dispensed into vials, degassed by 3 cycles of pure N ₂ exposure, scattered by briefly evacuating the chamber to 0.1 bar pressure, and the headspace contains pure N ₂ Sealed. After 8 weeks at 30 ° C. and 26 weeks at 5 ° C., samples were analyzed by SEC HPLC (described above) and after 37 weeks at 5 ° C. activity (chromogenic assay, described above). In addition, the same calcium concentration and 130 μg / mL sugar pegylated factor VIII, 0.2 M GuHCl, 18 mg / mL NaCl, 0.1 mg / mL polysorbate 80, 3 mg / mL sucrose, 0.055 mg / mL methionine and 1.5 mg / mL histidine, pH 6 .9 was used to build a rapid screening experiment. Samples were incubated for 24 hours at 5 ° C.

  Table 4 below lists the relative amount of free light chain and factor VIII activity obtained under different conditions.

  Increasing the calcium concentration from 3 to 10 mM at 5 ° C clearly results in better preservation of activity over 37 weeks and less free light chain formation over 26 weeks. This can be predicted after only 24 hours by assay with a chemical denaturant, but the accelerated stability at 30 ° C. has shown minimal margin. Further stabilization is obtained by bringing the calcium from 10 to 30 mM. Again, this is well predicted by chemical denaturation assays, but not by accelerated stability at 30 ° C.

130 μg / mL glycopegylated factor VIII (GP-BDD-FVIII), 0.4 M GuHCl, 18 mg / mL NaCl, 3.9 mM CaCl ₂ , 0.1 mg / mL polysorbate 80, 3 mg / mL sucrose, 0.055 mg / mL methionine and 1.5 The stabilizing effect of saccharides was investigated by preparing formulations with mg / mL histidine, pH 6.9 and different additional concentrations of saccharides. Samples were incubated for 24 hours at 5 ° C. and analyzed by SEC (described above). The relative areas of the free light chains obtained with the different stabilizers are listed in the following table 5 (Table 6).

  It can be seen that all three saccharides are efficient in stabilizing factor VIII liquid formulations.

  The effects of pH, NaCl concentration, sodium acetate (NaOAc) concentration, calcium chloride concentration and sucrose concentration on the liquid stability of glycopegylated factor VIII (GP-BDD-FVIII) in the presence of 0.35M GuHCl in multifactor experiments We investigated in.

  All samples contained 150 μg / mL GP-BDD-FVIII and 0.1 mg / mL polysorbate 80. The other components are shown in Table 6 below (Table 7). All of these formulations have calculated osmotic concentrations below 900 mOsm / L and do not take into account the content of GuHCl that is not part of the pharmaceutical formulation under development. Samples were incubated for 5 days at 5 ° C. and analyzed by SEC (described above). The relative area of the free light chain peak is also listed in this table.

It can be seen that in the absence of NaCl and NaOAc, the most free light chain formation is observed with 10 mM CaCl ₂ and without sucrose (Formulations 37, 40 and 43). Increasing the calcium concentration to 30 mM, adding sucrose to 300 or 600 mM, and adding NaCl or NaOAc all reduce the formation of free light chains. The slowest free light chain formation is observed with 200 mM NaOAc, 30 mM CaCl ₂ and 600 mM sucrose (formulations 30, 33 and 36).

Almost as good are samples with 200 mM NaCl, 30 mM CaCl ₂ and 600 mM sucrose (Formulations 12, 15 and 18). The difference between different values of pH is within experimental variation. These results confirm the stabilizing effect of 30 mM calcium and 300 or preferably 600 mM sucrose and also suggest that the complete absence of sodium is detrimental to the stability of Factor VIII in aqueous solution.

  To evaluate the optimal concentration of NaCl or NaOAc, experiments were conducted using urea as a chemical denaturant. Since GuHCl is a salt in itself, it can interfere with the determination of the optimum concentration of other salts.

All samples contained 100 μg / mL sugar pegylated FVIII (GP-BDD-FVIII), 1.6 M urea, 30 mM CaCl ₂ , 570 mM sucrose and 0.1 mg / mL polysorbate 80. The concentrations of histidine, NaCl and Na acetate (NaOAc) are listed in Table 7 below, along with the relative areas of free light chain after 96 hours at 5 ° C. The content of free LC was determined by SEC (described above). This table also lists osmostic concentrations calculated as described above and does not take into account the content of urea that is not part of the pharmaceutical formulation being developed.

  It can be seen that free light chain formation is most common in formulations that do not contain any sodium salt. Addition of 10 mM NaCl or 10 mM NaOAc improves stability. Further addition of NaCl up to 160 mM does not stabilize further, probably slightly destabilizing, while the addition of up to 160 mM NaOAc stabilizes slightly.

A number of formulations of GP-BDD-FVIII were prepared. All formulations contained 10 mM L-histidine, 0.02 mg / mL polysorbate 80, 0.5 mg / mL poloxamer 188, 0.37 mM L-methionine, 310 mM NaCl and 0.6 mg / mL sucrose and had a pH adjusted to 6.4. . The formulation contained approximately 250 U / mL GP-BDD-FVIII and different concentrations of CaCl ₂ . The solution was sterile filtered, dispensed into vials, degassed by 3 cycles of pure N ₂ exposure, and scattered by briefly evacuating the chamber to 0.1 bar pressure. The vial was closed with N ₂ in the headspace and incubated at 5 ° C. Samples were analyzed by molecular sieve chromatography (SEC, supra) after 4 and 8 weeks incubation. Table 8 below shows the relative area of this peak for different formulations.

An increase in the amount of free light chains, slower in 15 mM CaCl ₂ than 10 mM CaCl _2, even more slowly in 20 mM CaCl _2, it is seen still further slower in 30 mM CaCl _2. Variations in free light chain formation between 30, 45, 60 and 100 mM CaCl ₂ are within experimental uncertainty.

A series of preparations of factor VIII fused to the Fc domain (SEQ ID NO: 4) or albumin (SEQ ID NO: 5) were prepared. These proteins have an estimated long duration effect. The Fc fusion protein formulation contained approximately 200 U / ml Factor VIII derivative, 10 mM imidazole, pH 7.3, 0.1 mg / ml polysorbate 80, 0.5 M glycerol, 0.25 M NaCl and 0.6 M GuHCl. The albumin fusion protein formulation contained approximately 500 U / ml Factor VIII derivative, 10 mM imidazole, pH 7.3, 0.1 mg / ml polysorbate 80, 0.5 M glycerol, 0.25 M NaCl and 0.6 M GuHCl. In addition, formulations contained sucrose and CaCl ₂ different concentrations. The formulation was incubated for about 24 hours at 5 ° C. and analyzed by molecular sieve chromatography (SEC, supra). The following Table 9 lists the CaCl ₂ and sucrose concentrations and the measured relative light chain areas.

  It can be seen that accelerated assays are also applicable to these factor VIII derivatives. It can also be seen that a calcium concentration of 30 mM and a sucrose concentration of 500 mM have a beneficial effect on the liquid stability of the factor VIII derivative.

A series of formulations of BDD-FVIII was prepared. All formulations contained in common the following components: about 2500 IU / ml BDD-FVIII, 310 mM NaCl, 20 mM histidine, 6 mg / ml sucrose, 0.11 mg / ml methionine, 0.2 mg / ml polysorbate 80 and 0.4 M. Guanidine hydrochloride. The formulation further contained different concentrations of calcium chloride in the range of 3-100 mM. The formulation was incubated at 5 ° C. for about 24 hours and analyzed by molecular sieve chromatography (SEC, supra). The following Table 10 lists the relative integrals of different concentrations of CaCl ₂ light chain.

  It can be seen that BDD-FVIII is stabilized by increasing the concentration of calcium above 10 mM, with values of 30-100 mM being almost equally effective.

  A series of formulations of BDD-FVIII was prepared. All formulations contained in common the following components: about 500 IU / ml BDD-FVIII, 310 mM NaCl, 7 mM histidine, 2 mg / ml sucrose, 0.04 mg / ml methionine, 0.07 mg / ml polysorbate 80 and 0.6 M. Guanidine hydrochloride. The formulation further contained different concentrations of calcium chloride in the range of 1-30 mM. Samples were incubated for 4 days at 5 ° C. and assayed for factor VIII activity by a chromogenic assay (Coatest® SP FVIII, supra). The measured activities are listed in the following table.

  It can be seen that accelerated stability screening can also be performed using biological activity assays.

A series of formulations of BDD-FVIII was prepared. All formulations contained in common the following components: about 2000 IU / ml BDD-FVIII, 0.6 M GuHCl, 30 mM CaCl ₂ , 570 mM sucrose, 0.1 mg / ml polysorbate 80, 40 mM NaCl and 10 mM histidine. The pH value varied between 5.5 and 7.2. All formulations had a calculated osmotic concentration of about 743 mOsm / L without taking into account the content of GuHCl that was not part of the pharmaceutical formulation being developed. The formulation was incubated at 5 ° C. for 3 days and analyzed by molecular sieve chromatography (SEC, supra). The following table lists the relative integrals of light chains with different pH values.

  It can be seen that a pH value around 6.5 is most advantageous.

Two formulations of GP-BDD-FVIII were prepared. Both formulations contained approximately 290 U / ml GP-BDD_FVIII, 10 mM histidine, 30 mM CaCl ₂ , 1.5 mg / ml pluronic F68, 600 mM sucrose and had a pH adjusted to 6.7. One formulation did not contain NaCl and the other contained 78 mM NaCl. The formulation without NaCl had a calculated osmolarity of about 700 mOsm / L, and the formulation with 78 mM NaCl had a calculated osmolarity of about 845 mOsm / L. The solution is sterile filtered, dispensed into vials, degassed by 3 cycles of pure N ₂ exposure, scattered by briefly evacuating the chamber to 0.1 bar pressure, and the headspace is filled with pure N ₂ Was sealed. Samples were incubated for 52 weeks at 5 ° C or -80 ° C and activity was measured by chromogenic assay. The table below lists the results.

  It can be seen that the activity is very well preserved in both formulations and that the sample stored at 5 ° C showed essentially the same activity as the reference stored at -80 ° C.

Eight types of GP-BDD-FVIII formulations were prepared. All formulations contained about 300 U / ml GP-BDD-FVIII, 30 mM CaCl ₂ , 0.1 mg / ml polysorbate 80 and 500 mM sucrose. The formulations contained different concentrations of histidine, NaCl, Na acetate and were adjusted to different pH values as detailed in the table below. The solution is sterile filtered, dispensed into vials, degassed by 3 cycles of pure N ₂ exposure, scattered by briefly evacuating the chamber to 0.1 bar pressure, and the headspace contains pure N ₂ Sealed. Samples were incubated at 5 ° C. or −80 ° C. for 32 weeks and the activity was measured by the color development method. The results are also listed in the table.

  It can be seen that the sample stored at 5 ° C shows almost the same activity as the reference stored at -80 ° C, and the activity is particularly well preserved at pH 6.4-6.9.

Eight types of GP-BDD-FVIII formulations were prepared. All formulations contained approximately 290 U / ml GP-BDD_FVIII, 0.3 mM methionine, 30 mM CaCl ₂ , 0.1 mg / ml polysorbate 80 and 10 mM histidine and had a pH of 6.5. The formulations contained different concentrations of sucrose and NaCl as detailed in the table below. The solution is sterile filtered, dispensed into vials, degassed by 3 cycles of pure N ₂ exposure, scattered by briefly evacuating the chamber to 0.1 bar pressure, and the headspace contains pure N ₂ Sealed. Samples were incubated at 5 ° C. or −80 ° C. for 32 weeks and the activity was measured by the color development method. The results are also listed in the table.

  It can be seen that, except for the formulation containing 9 mM sucrose, the samples stored at 5 ° C. show approximately the same activity as the reference stored at −80 ° C. Obviously, a higher sucrose concentration imparts high stability to the formulation, even at NaCl concentrations much lower than those previously described for stable liquid Factor VIII formulations.

Four formulations of full length factor VIII [Kogenate ™] were prepared. All formulations contained 500 IU / ml Factor VIII, 0.6 M GuHCl, 20 mM histidine, 38 mM NaCl, 0.1 mg / ml polysorbate 80, pH 6.9. In addition, the formulations contained different amounts of CaCl ₂ and sucrose. The formulation was incubated for about 24 hours at 5 ° C. and assayed for free LC content by SEC chromatography. The results are listed in the table below.

  It can be seen that full-length factor VIII is also stabilized against dissociation of the light chain from the heavy chain by increasing calcium and sucrose concentrations, and is particularly stabilized when both calcium and sucrose concentrations are increased.

Six formulations of GP-BDD-FVIII were prepared. All formulations contained 30 mM CaCl ₂ and 0.055 mg / ml L-methionine. The other components are as listed in the table below. The solution is sterile filtered, dispensed into vials, degassed by 3 cycles of pure N ₂ exposure, scattered by briefly evacuating the chamber to 0.1 bar pressure, and the headspace contains pure N ₂ Sealed. The formulations were stored for 9 months at -80 ° C or 5 ° C and assayed for activity by chromogenic assay. The results are listed in the table.

  It can be seen that the activity is well preserved after 5 ° C for 9 months.

Two formulations of BDD-FVIII were prepared. Both formulations, 30 mM CaCl _2, 10 mM histidine, containing 570mM sucrose, 78 mM NaCl, 0.1 mg / ml polysorbate 80 and 0.055 mg / ml L-methionine, having a pH adjusted to 6.7. The formulations had different concentrations of Factor VIII as listed in the table below. The solution is sterile filtered, dispensed into vials, degassed by 3 cycles of pure N ₂ exposure, scattered by briefly evacuating the chamber to 0.1 bar pressure, and the headspace contains pure N ₂ Sealed. The formulations were stored for 9 months at -80 ° C or 5 ° C and assayed for activity by chromogenic assay. The results are listed in the table.

  It can be seen that the activity is well preserved after 5 ° C for 9 months.

  While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes and equivalents will occur to those skilled in the art. Accordingly, it is to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims (28)

A factor VIII molecule;
A calcium salt with a concentration exceeding 10 mM,
A liquid aqueous formulation of coagulation factor VIII comprising a sugar and / or polyol at a concentration of at least 100 mM and having a pH of 5.5 to 7.5. A factor VIII molecule;
A calcium salt at a concentration of at least 15 mM;
2. A liquid aqueous formulation of coagulation factor VIII according to claim 1, comprising a saccharide and / or polyol at a concentration of at least 100 mM and having a pH of 5.5 to 7.5.   Calcium salt is 15-100 mM, or 15-80 mM, or 15-60 mM, or 15-45 mM, or 20-100 mM, or 20-80 mM, or 20-60 mM, or 20-45 mM, or 20-40 mM, or 25 3. A formulation according to claim 1 or 2 present at a concentration of ~ 35mM.   The preparation according to any one of claims 1 to 3, wherein the calcium salt is calcium acetate, calcium lactate, calcium benzoate or calcium chloride, or a mixture of two or more thereof.   The formulation according to claim 4, wherein the salt is calcium chloride.   6. The formulation according to any one of claims 1 to 5, further comprising a sodium salt at a concentration of at least 5 mM.   The sodium salt is present in any one of claims 1-6, wherein the sodium salt is present at a concentration of 5-500 mM, or 15-200 mM, or 15-150 mM, or 15-100 mM, or 50-150 mM, or 5-50 mM. Formulation.   The formulation according to claim 6 or 7, wherein the sodium salt is sodium chloride or sodium acetate, or a mixture thereof.   The preparation according to any one of claims 1 to 8, wherein the polyol is a monosaccharide or disaccharide, or a sugar alcohol, or a combination thereof.   10. A formulation according to claim 9, wherein the mono- or disaccharide and / or sugar alcohol is selected from the group of sucrose, sorbitol, glycerol, raffinose, stachyose, mannitol, sorbitol, or mixtures thereof.   Monosaccharide or disaccharide and / or sugar alcohol is at least 100 mM, or at least 200 mM, or 100-1800 mM, or 300-1800 mM, or 100-1500 mM, or 200-1800 mM, or 200-1500 mM, or 100-1000 mM, or 11. A formulation according to claim 9 or 10 present at a concentration of 200-1000 mM, or 300-1000 mM, or 200-800 mM, or 300-800 mM, or 400-800 mM, or 500-800 mM, or 500-700 mM.   A sucrose in a concentration of 50-600 mg / mL, or 100-600 mg / mL, or 100-450 mg / mL, or 150-450 mg / mL, or 150-300 mg / mL. The preparation according to item.   13. A formulation according to any one of claims 1 to 12, containing sorbitol at a concentration of at least 400 mM.   14.The formulation of claim 13, wherein the sorbitol is present at a concentration of 100-800 mg / mL, or 100-650 mg / mL, or 150-650 mg / mL, or 150-500 mg / mL, or 150-250 mg / mL. .   The formulation according to any one of claims 1 to 14, wherein the calculated osmotic pressure concentration of the formulation is at most 1500 mOsm / L, 1200 mOsm / L, 1000 mOsm / L or 900 mOsm / L.   16. A formulation according to any one of claims 1 to 15, having a pH of 5.5 to 7.5, or 6.0 to 7.0, or 6.3 to 6.7.   The preparation according to any one of claims 1 to 16, wherein the factor VIII molecule is a recombinant full-length FVIII or a recombinant B-domain truncated FVIII.   The formulation according to any one of claims 1 to 16, wherein the factor VIII molecule is an FVIII derivative or an FVIII analog.   19. The formulation of claim 18, wherein the factor VIII molecule is a pegylated FVIII, or an FVIII fusion protein such as albumin fusion FVIII or Fc region fusion FVIII.   20. The formulation of claim 19, wherein the factor VIII molecule is glycopegylated B domain truncated FVIII.   A double chain consisting of a heavy chain-linker sequence (A1-a1-A2-a2-L) and a light chain sequence (a3-A3-C1-C2), in which FVIII molecules are joined together by non-covalent interactions B domain truncated FVIII molecule, the linker (L) is a 20 amino acid residue linker sequence (SFSQNSRHPSQNPPVLKRHQ) (SEQ ID NO: 3), heavy chain (A1-a1-A2-a2) and light chain (a3 The preparation according to any one of claims 1 to 17, wherein -A3-C1-C2) corresponds to the sequences represented by amino acid numbers 1 to 740 and 1649 to 2332 of SEQ ID NO: 1, respectively.   A double chain consisting of a heavy chain-linker sequence (A1-a1-A2-a2-L) and a light chain sequence (a3-A3-C1-C2), in which FVIII molecules are joined together by non-covalent interactions B domain truncated FVIII molecule, the linker (L) is a 20 amino acid residue linker sequence (SFSQNSRHPSQNPPVLKRHQ) (SEQ ID NO: 3), heavy chain (A1-a1-A2-a2) and light chain (a3 -A3-C1-C2) corresponds to the sequences represented by amino acid numbers 1 to 740 and 1649 to 2332 of SEQ ID NO: 1, respectively, and one or more PEG groups are present in the linker sequence (SEQ ID NO: 3). 21. A formulation according to any one of claims 1 to 20 which is bound to FVIII polypeptide via a located glycan. (i) providing one or more liquid preparations containing Factor VIII to be tested;
(ii) adding a protein denaturant to the liquid formulation and incubating the resulting solution for a predetermined period;
(iii) analyzing the incubated solution of (ii) for the presence of dissociated factor VIII;
(iv) selecting one or more formulations having a desired low level of dissociated factor VIII, and a method for optimizing a liquid formulation of coagulation factor VIII. (i) providing one or more liquid preparations containing Factor VIII to be tested;
(ii) adding a protein denaturant to the liquid formulation and incubating the resulting solution for a predetermined period;
(iii) analyzing the incubated solution of (ii) for the presence of dissociated factor VIII;
(iv) selecting a one or more formulations having the desired low level of dissociated factor VIII, and identifying a stable liquid formulation of factor VIII.   The method according to claim 23 or 24, wherein the protein denaturant is guanidinium chloride or urea.   26. The method according to any one of claims 23 to 25, wherein the factor VIII molecule is recombinant full length FVIII or recombinant B domain truncated FVIII.   26. A method according to any one of claims 23 to 25, wherein the factor VIII molecule is an FVIII derivative or an FVIII analog.   28. The method of claim 26 or 27, wherein the factor VIII polypeptide is glycopegylated B domain truncated FVIII.